Abstract
The cyanobacterial circadian clock oscillator is composed of three clock proteins--KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC(1mer)) and formed a complex with KaiB. The addition of ATP plus Mg(2+) (Mg-ATP), but not that of ATP only, to the KaiB-KaiC(1mer) complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC(1mer) complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg(2+) (Mg-ATP), KaiC became a homohexameric ATPase (KaiC(6mer)) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC(6mer) lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC(6mer) is required for formation of the KaiB-KaiC(6mer) complex. KaiC(6mer) that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC(6mer) with bound ADP-Pi.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Diphosphate / chemistry
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Adenosine Diphosphate / metabolism
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Adenosine Triphosphatases / chemistry
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Adenosine Triphosphate / chemistry
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Adenosine Triphosphate / metabolism
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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Cations, Divalent
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Circadian Clocks / genetics*
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Circadian Rhythm Signaling Peptides and Proteins / chemistry
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Circadian Rhythm Signaling Peptides and Proteins / genetics*
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Circadian Rhythm Signaling Peptides and Proteins / metabolism
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Cyanobacteria / genetics*
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Cyanobacteria / metabolism
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression Regulation, Bacterial*
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Magnesium / chemistry
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Magnesium / metabolism
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Phosphorylation
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Protein Multimerization
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Protein Structure, Tertiary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
Substances
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Bacterial Proteins
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Cations, Divalent
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Circadian Rhythm Signaling Peptides and Proteins
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KaiA protein, cyanobacteria
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KaiB protein, cyanobacteria
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KaiC protein, cyanobacteria
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Recombinant Proteins
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Adenosine Diphosphate
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Adenosine Triphosphate
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Adenosine Triphosphatases
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Magnesium
Grants and funding
This work was supported by grants (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to MI and RM and the Sasakawa Scientific Research Grant from the Japan Science Society to AN. RM was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.